DESCRIPTION(provided by the applicant): Systemic lupus erythematosus (SLE) is a chronic multisystem disease that is associated with deficiency of C4A but not the C4B isotype of human complement. While the etiology is uncertain two models exist addressing this association. The first or clearance hypothesis suggests that pathogenesis results from inadequate clearance and processing of immune complexes. Normally, processing of immune complexes requires the activation and covalent binding of C4 (C4b) and C3 (C3b) thus preventing aggregation and precipitation of antibody-antigen complexes. The soluble complexes then bind to C4b/C3b receptors (CD35) expressed on erythrocytes and are transported from the circulation to the mononuclear phagocytic system for normal disposal. The second, or negative selection model, proposes that complement plays a critical role in immune tolerance by eliminating self-reactive B-cells. Under this model anergy is maintained by immune complex activation and coupling of C4b to self-antigens released from necrotic or apoptotic cells thus providing a ligand for CD35 on the surface of follicular dendritic-like stromal cells. Emerging immature B-cells then encounter self-antigen in the presence of C4b and C3d and are tolerized or eliminated. Our overall hypothesis is that structural differences between C4 isoforms differentially affect immune complex clearance and/or the balance between self-reacting B-cells and the normal antibody response through differences in avidity with CD35. Specifically, that the 1101 Proline in C4A contributes to a tertiary structure that increases avidity to CD35, as compared with C4B, which has a 1101 Leucine. Furthermore, that the 1103 Prolines of both isoforms are involved in maintaining the CD35 binding site. Therefore, switch mutants at position 1101 and substitution mutants at 1103 will be generated by site-directed mutagenesis and expressed using a modified baculovirus expression system. The proposed research will examine the effect of C4 isotype on binding of immune complexes to CR1 and release by factor I. Binding avidities will be determined using surface plasmon resonance and also by using human erythrocytes and radiolabeled dimers of mutant and wild-type C4b, and mixed C4b-C3b dimers. Structural analyses performed using several spectroscopic methods. [unreadable] [unreadable]